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CN1094445A - F1K-1 as vascular endothelial growth factor receptor - Google Patents

F1K-1 as vascular endothelial growth factor receptor Download PDF

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Publication number
CN1094445A
CN1094445A CN93115345A CN93115345A CN1094445A CN 1094445 A CN1094445 A CN 1094445A CN 93115345 A CN93115345 A CN 93115345A CN 93115345 A CN93115345 A CN 93115345A CN 1094445 A CN1094445 A CN 1094445A
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flk
vegf
cell
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expression
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CN1173991C (en
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阿克塞尔·乌尔里希
维尔纳·里绍
比吉特·米劳尔
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Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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Abstract

The present invention relates to the use of ligands for the F1K-1 receptor to modulate angiogenesis and vasculogenesis. The present invention is based, in part, on the demonstration that F1K-1 tyrosine kinase receptor expression is associated with endothelial cells and the identification of Vascular Endothelial Growth Factor (VEGF) as a high affinity ligand for F1K-1. These results indicate the major role of F1K-1 in the signaling system during both vascularisation and vascularization. Discloses the genetic engineering design of a host cell expressing F1K-1 and the application of the expressed F1K-1 in evaluating and screening VEGF medicines and analogues related to F1K-1 regulation through agonist or antagonist activity. The invention also relates to the use of F1K-1 ligands, including VEGF agonists and antagonists, in the treatment of diseases, including cancer, by modulating angiogenesis and vasculogenesis.

Description

F1K-1 as vascular endothelial growth factor receptor
The part that the present invention relates to the Flk-1 acceptor is to regulating the purposes of vascularization and vasculation.The present invention is based on partly that the Flk-1 tyrosine kinase receptor is expressed the proof relevant with endotheliocyte and as the discriminating of the vascular endothelial growth factor (VEGF) of the high-affinity part of Flk-1.These results have pointed out the main effect of Flk-1 in signalling system during vasculation and the vascularization.The Flk-1 that the invention discloses the engineering of the host cell of expressing Flk-1 and expressed is to estimating and screen the VEGF medicine related in the Flk-1 adjusting and the purposes of analogue, and described adjusting is carried out with agonist or antagonistic activity.
The present invention also relates to the Flk-1 part, this comprises VEGF agonist and antagonist, treating disease by regulating vasculation and vascularization, comprises the effect in the cancer.
Receptor tyrosine kinase comprises a big nation with horizontal membrane receptor that collapses of various bioactive polypeptide growth factor.Their inherent Tyrosylprotein kinase functions part in conjunction with the time be activated, this causes the phosphorylation of this receptor and many cells material, and forms various kinds of cell reaction (Ullrich A.and Schlessinger, J. subsequently, 1990, Cell 61: 203-212).
Receptor tyrosine kinase cDNA, the mouse cell group that is referred to as Flk-1 (Flk-1) and is by enrichment green blood stem and progenitor cell clones.This receptor be considered to the green blood stalk cell upgrade relevant (Matthews et al., 1991, Proc.Natl.Acad.Sci.USA 88: 9026-9030).Flk-1 clone's sequential analysis has shown the C-kit subfamily with receptor kinase, particularly with sizable homology of Flt gene product.These acceptors all have the zone, a kind of extracellular that contains immunoglobulin (Ig) shape structure jointly.
The formation of blood vessel and expansion, or vasculation and vascularization, respectively in various physiological processes, in fetal development, wound healing, neomorph and female reproduction process, as playing an important role in the growth of the folliculus in the corpus luteum between the onset of ovulation and the back placenta growth of becoming pregnant.Uncontrolled vascularization may be pathologic, as depends on the growth of solid tumor of the vascularization of growth.
Vascularization comprises hyperplasia, migration and the infiltration of vascular endothelial cell, and is likely by polypeptide growth factor and regulates.Differentiated that some has stripped endothelial cell growth and promotes active polypeptide.Example comprises acid and alkalescence or fibroblast growth factor, vascular endothelial growth factor and placenta growth factor.Although four kinds of distinct acceptors of existing FGF family different members by characterization, did not wherein also have a kind of the report to be expressed in blood vessel in vivo.
Although FGF seems it is cytokinin to a large amount of different cell types, but reported VEGF recently is a kind of specific disruption element (Ferrara, N.and Henzel, W.J. of endotheliocyte, 1989, Biochem.Biophys Res.Comm.161: 851-858).Recently, fms class tyrosine acceptor, flt shown the affinity that has VEGF (Devries, C.et al., 1992, Science 255: 989-991).
The present invention relates to use the part of Flk-1 acceptor to regulating the purposes of vascularization and vasculation.The present invention partly is based on such discovery: the Flk-1 tyrosine kinase receptor is expressed and based on the vascular endothelial growth factor of having differentiated as the high-affinity part of Flk-1 (VEGF) at endothelial cell surface.Shown the vital role of Flk-1 in these processes at endotheli ocytosis and transporting action during vascularization and the vasculation.The present invention describes with the example of mouse Flk-1, yet these principles can be used for other species, comprise the mankind.
Attempt suppresses the interactional medicament of Flk-1/VEGF and can be used for suppressing tumor growth.VEGF and/or VEGF agonist can be in order to promote wound healing.The present invention relates to be predefined for the expression system of producing expression Flk-1 acceptor Flk-1 albumen and/or clone.The proteic expression of soluble recombinant chou Flk-1 can be used for screening the peptide storehouse that suppresses the interactional molecule of Flk-1/VEGF.The engineering cell system of expressing Flk-1 on their surfaces can be advantageously in order to jig and to differentiate VEGF agonist and antagonist.
The comparison of Fig. 1 .Flk-1 aminoacid sequence and relevant RTK.Flk-1 and human body KDR and rat Tk 1The comparison of the aminoacid sequence of-C.The paragraph zone of all three kinds of acceptor known arrays is compared but only shows difference with the Flk-1 sequence.
The Northern engram analysis of Fig. 2 .Flk-1 genetic expression.
(A) expression of Flk-1RNA in 9.5-18.5 days mice embryonics.In per pass, analyzed the total RNA sample (10mg) that obtains by whole mice embryonics.Indicated the position of 28s and 18s ribosome-RNA(rRNA).(B) with from the back four days brain kapillary fragment of birth relatively Flk-1mRNA birth back four days with adult brain in expression.On per pass, be loaded with 1mgPoly(A +) RNA.5 ' the 2619bp that uses Flk-1cDNA is as probe.Contrast hybridization with GAPDH cDNA probe has been shown in the grid of bottom.
Fig. 3. volume Flk-1 expression of gene in embryonic tissue.The in situ hybridization analysis that Flk-1 expresses in 14.5 days mice embryonics.(A) by all with the antisense probe of 35s mark (5 ' 2619bp) embryos' of hybridizing parallel light field illumination of vowing the shape zone.(B) the details in a play not acted out on stage, but told through dialogues illumination of same area.(C) with the contrast hybridization of the adjacent area of meaningful probe.Abbreviation: Ao, aorta; At, the atrium; L, lung; Li, liver; Ma, mandibular bone; Mn, meninx; Ms, midbrain; T, akrencephalon; V, ventricle; Vt, vertebra.
Fig. 4. the expression of Flk-1 RNA among the embryonic organ is limited to specific cells.The Flk-1 rna expression in 14.5 days mice embryonics that high power is amplified.(A) heart district that surveys with the antisense probe of 35s mark.(B) with the adjacent area of meaningful probe hybridization.(C) with the part aorta wall shown in the cell levels.Endothelial layer is indicated by an arrow.(D) lung of surveying with the Flk-1 antisense probe.(E) with the contrast hybridization of the adjacent area of meaningful probe hybridization.Abbreviation: At, atrium; B, segmental bronchus; Ed, endothelial layer; En, endocardium; L, lung; Li, liver; Lu, aorta lumen; M1, muscle; My, cardiac muscle.
Fig. 5. the Flk-1 genetic expression in developmental mouse brain.The in situ hybridization analysis of the Flk-1 genetic expression in the brain of different developmental phases.All zones all uses the Flk-1 antisense probe to survey.(A) the arrow shape district of 11.5 days mice embryonic akrencephalon.Represent to express the independent blood vessel of Flk-1 by arrow, this blood vessel is developed into neural outer by meninx.(B) 14.5 days embryos and (C) the arrow shape zone of back four days brains of birth.Shown is the midbrain zone.Branched hair tubule and the blood vessel of Flk-1 have been pointed out to express by arrow.(D) the arrow shape district of adult brain; Show the zone of midbrain.Pointed out to express the cell of Flk-1 by arrow.Abbreviation: M, meninx; V, ventricle.
Fig. 6. the expression of the Flk-1 in the choroid plexus of the brain of growing up.(A) with the details in a play not acted out on stage, but told through dialogues illumination of the adult rats brain choroid plexus of Flk-1 antisense probe hybridization.(B) to exchange the choroid plexus shown in amplifying.Arrow has pointed out to show the single cell of strong Flk-1 expression.Abbreviation: Cp, choroid plexus; E, ependyma; Ep, epithelial cell; V, ventricle.
Fig. 7 expresses Flk-1 in renal glomerulus.(A) with the parallel arrow shape district of the back four days kidneys of birth of Flk-1 antisense probe hybridization.Pointed as arrow, hybridization signal is accumulated in the renal glomerulus.(B) the arrow shape district of the adult kidney of surveying with Flk-1 with the contrast of meaningful probe adjacent area hybridization (C).Arrow has been pointed out renal glomerulus.(D) the adult renal glomerulus of high power amplification.(A) and the arrow (D) pointed out to be arranged in cell in the chain in the nearly bead zone of expressing Flk-1.
Fig. 8. the in situ hybridization analysis of the expression of the Flk-1 in embryo and the embryo outside organization in early days.(A) in arrow shape district with 8.5 days mice embryonics in the parent cast of Flk-1 probe.Arrow has been pointed out the endothelium of the parent blood vessel of strong expression Flk-RNA.(C) high power of 9.5 days mice embryonic yolk sac and trophectoderm is amplified.(D) high power of blood island is amplified.Abbreviation: A, allantois; Bi, blood island; Bv, parent blood vessel; D, cast; En, the endodermis of yolk sac; M, a matter; Ms, the mesoderm of yolk sac; NF, neural fold; T, trophoderm; Y, yolk sac.
Fig. 9 .Flk-1 is a kind of vegf receptor.(A) 125The COS cell of I-VEGF and transient expression Flk-1 acceptor is crosslinked, and uses 125I-VEGF will contrast cell culture in 4 ℃ and spend the night, and use salt solution (PBS) washed twice of phosphate buffered then and be exposed among the linking agent DSS of 0.5mM in PBS through 1 hour in 4 ℃.With cytolysis, immunoprecipitation goes out the Flk-1 acceptor, and by the polyacrylamide gel electrophoresis analysis, with after the radioautogram analysis.Indicated the molecular dimension mark with Kb.(B) 125The particular combination of the COS cell of I-VEGF and expression Flk-1.Dividing the COS cell of removing transient expression F1k-1 also to be suspended in again from flat board connects the medium (DMEM, 25mM Hepes, 0.15% gelatin).Containing 2 * 10 5Cell, 15000cpm 125I-VEGF is to be connected 90 minutes in 15 ℃ in the medium of 0.5ml with the cumulative volume of the unmarked part of prescribed concentration.Wash these cells twice and on gamma counter, count with PBS/0.1%BSA.
The automatic phosphorylation of VEGF inductive of Figure 10 .Flk-1.In the DMEM that contains 0.5% tire calf serum, make the COS cell of transient expression Flk-1 acceptor as noted and contrasted cell hungry 24 hours, stimulated 10 minutes with VEGF then.These cells are dissolved, and the immunoprecipitation with the Flk-1 acceptor polyclonal antibody that resists its C not hold separates with polyacrylamide gel electrophoresis, transfers to soluble cotton.Survey trace with anti-phosphotyrosine antibody (5B2).By using horseradish-peroxidase link coupled secondary antibodies and BCLTM(Amersham) this protein band of detection test visual detection.
Figure 11. the nucleotide sequence of the Flk-1 of mouse.
Figure 12. the plasmid figure of retroviral vector member.PLXSN Flk-1 TMc1.1 and pLXSN Flk-1 TMc1.3 contain Flk-1 amino acid/11 to 806.PNTK-cfms-TM contains 541 n terminal amino acids of c-fms.
Figure 13. the trans-dominant-feminine gender by Flk-1 suppresses C 6The inhibition of glioblastoma knurl growth.C 6Cell or separately transplanted or transplant with the cell that produces virus.In each picture, pointed out cell count.Used the virocyte system of two kinds of different generations: a kind of expression Flk-1 TM(trans-dominant feminine gender) mutant and the negative c-fms mutant (c-fms TM) of a kind of expression trans-dominant in contrast.When the first time, tumour occurred, measured tumor size to obtain growth curve in every 2-3 days.Four mouse are represented each group.
Figure 14. the trans-dominant-feminine gender by Flk-1 suppresses the glioblastoma tumor growth.C 6Cell or separately transplanted or with the virocyte co-transplantation that produces.Pointed out cell count in each picture.Used the clone of two kinds of different generation viruses: a kind of expression Flk-1 TM(trans-dominant-feminine gender) mutant and a kind of as a comparison expression trans-dominant-negative c-fms mutant (c-fms TM).When the first time, tumour occurred, measured gross tumor volume to obtain growth curve in every 2-3 days.Every group represented by four mouse.
The part that the present invention relates to the Flk-1 acceptor is to regulating the purposes of vascularization and/or vasculation.The present invention also relates to estimate and screen the expression of the Flk-1 of VEGF medicine and analogue, described medicament and analogue are relevant with receptor activation, adjusting and uncoupling.The regulatory gene of these Flk-1 can be used in treatment.For example, the agonist of VEGF can be used for like an elephant the such process of wound healing, and on the contrary, the antagonist of VEGF can be used for treatment and depends on vasculation and growing tumors.
The present invention partly is based on by showing that Flk-1 is the in situ hybridization of the specific RTK of a kind of endotheliocyte and the result of Northern engram analysis gained.In addition, it is a kind of high-affinity receptor to dimension endothelial tube somatomedin (VEGF) that crosslinked test has demonstrated Flk-1, and this shows that F1k-1 plays a decisive role to angioblastic growth and differentiation and endothelial cell growth subsequently during vasculation and vascularization.
The present invention is also based on such discovery: the biological activity of the endogenous wild-type Flk-1 that the expression of the trans-dominant of Flk-1 molecule-negative mutant forms can suppress.This paper has described these tests, in these trials with tumour cell with produce the Flk-1 acceptor retroviral cytomixis of recombinant chou of encoding that will be truncate and be injected in the mouse.In the mouse of the Flk-1 acceptor of expressing truncate shape, observe the inhibition of vascularization and injected growth of tumour cell.The expression of F1k-1 molecule trans-dominant-negative form can be used for treating because the disease that the aberrant angiogenesis hyperplasia causes is grown as rheumatic arthritis, retinopathy and solid tumor.
Explain as the embodiment that hereinafter works, used polymerase chain reaction (PCR) method to separate the novel receptor Tyrosylprotein kinase of specially after transplanting, expressing in embryo and the endotheliocyte.Found a kind of like this clone, a RTK is encoded, it have similar and previous differentiate clone identical sequence homology (Matthews et al. by isolated cDNA in the population of cells of enrichment hemopoietic cell and specified fetus liver kinases-1(Flk-1), 1991, Proc.Natl.Acad Sci.U.S.A.88: 9026-9030) (Figure 11).
Clear for what discuss, the Flk-1 with mouse is example explanation the present invention in the paragraph below.Yet these principles can be applied to the clone similarly and express other species, comprise human Flk-1.
1.Flk-1 encoding sequence
The nucleotide coding sequence of mouse Flk-1 gene and the aminoacid sequence of deriving are described in Figure 11 (SEQ.ID NO.1) and open recently (Matthews et al., 1991, Proc.Natl.Acad.Sci.U.S.A, 88: 9026-9030).In the Mammals, comprise that the human proteic nucleotide sequence of Flk-1 or its function equivalent can be used for producing the recombinant chou molecule that causes Flk-1 to express; This acceptor will be referred to as " Flk-1 " hereinafter, and what species be produced irrelevant by.
In a specific embodiment disclosed herein, isolate mouse Flk-1 gene (Hanks et al., 1988) by the polymerase chain reaction (PCR) that uses two bases to carry out in the degeneracy oligonucleotide primer storehouse that inner height conservative sequence in receptor tyrosine kinase district designs.The DNA in the λ gt10 cDNA storehouse that use is made by 8.5 days mice embryonics is as template.In parallel mode, use the RTK cDNA sequence of similar primer with isolated capillary endothelial cells in the afterwards 4-8 days mouse brains of enriching freely to be born.This is the time of brain endothelial cell hyperplasia maximum.Two kinds of methods all produce disclosed fetus liver RTK recently, the cDNA sequence (Matthews et al., 1991) of Flk-1 coding.According to amino acid identity, this acceptor is a member (Ullrichand Schlessinger) in the RTK III type subclass, and RTK contains immunoglobulins replica (Fig. 1) in its zone, extracellular.
The present invention also relates to by other species, comprise human isolating Flk-1 gene, wherein have the Flk-1 activity.This paper is with VEGF or segmental those acceptor definition Flk-1 member of family of binding peptide.80% homology on the aminoacids content of these acceptor susceptible of proofs in the actual range of dna sequence dna.Use radiolabeled mouse Flk-1 cloned sequence, under the condition that lowers intensity, can screen phage cDNA storehouse.In other words, mouse Flk-1 sequence can be used to the oligonucleotide sequence that designs the degeneracy that can be used as the PCR probe or finish degeneracy or screen phage cDNA storehouse.Strategy based on polymerase chain reaction can be used for cloning human body Flk-1.Can be with the primer in reacting as PCR corresponding to two kinds of degeneracy oligonucleotide storehouses that keep characteristic between mouse Flk-1 and the receptor tyrosine kinase.The template of this reaction is the cDNA that the reverse transcription by the mRNA that is produced by the clone of known expression human body Flk-1 or tissue obtains.The PCR product can be represented the Flk-1 sequence to guarantee the sequence that quilt enriches by subclone and sequencing.Can use this PCR fragment, clone to isolate total length Flk-1 cDNA by fragment and screening phage cDNA storehouse that radio-labeling amplifies.On the other hand, can use this fragment that is labeled with the screening-gene storehouse.For commenting spendable clone's scheme, referring to for example Maniatis, 1989, Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y.; And Ausubel et al., 1989, Current Protocols in Molecular Biology, (Green Publishing Associates and Wiley Interscience, N.Y.).
Also can be by in mammalian expression vector, as contain can be when being transferred to the COS cell height duplicate construction cDNA storehouse among the PCDNAI in SV40 source of replication sequence that numerical table reaches plasmid and finish the separation of human body Flk-1cDNA.Available serial of methods detects the expression of the Flk-1 on the COS of transfection cell surface, and this comprises the part of applying marking, as VEGF or the VEGF antagonist with radio-labeling, fluorescent mark or enzyme labelling.Can be by cells transfected be carried out the FACS(fluorescence activated cell sorter) classification comes enrichment to express the cell of human body Flk-1.
According to the present invention, can in proper host cell, use the Flk-1 nucleotide sequence that peptide fragment, Flk-1 fusion rotein or its function equivalent of Flk-1, Flk-1 are encoded to produce the recombinant DNA molecules of the expression that causes Flk-1 albumen or its function equivalent.On the other hand, in Nucleotide cross experiment, Southern and Northern engram analysis etc., also can use the nucleotide sequence of hybridizing with Flk-1 sequence position.
Because the intrinsic degeneracy of gene-code can be used amino acid sequences encoded other dna sequence dna that will be equal on substantially the same or the function in practice of the present invention, with clone and expression Flk-1 albumen.These dna sequence dnas be included under the stringent condition can with those sequences of mouse Flk-1 sequence hybridization.
The dna sequence dna that can change used according to the present invention comprises the disappearance of different nucleotide residues, additional or replacement, and the result forms the sequence of the gene product coding that will be equal on identical or the function.This can have the disappearance of amino-acid residue, additional or replacement this gene product in Flk-1 sequence scope, the result causes quiet variation, thereby produces the Flk-1 that is equal on the function.Can carry out these amino acid whose replacements according to the similarity of polarity, electric charge, solubleness, hydrophobicity, wetting ability and/or the amphipathic characteristic aspect of related residue.For example, the amino acid of bear electricity comprises aspartic acid and L-glutamic acid; The amino acid of lotus positive electricity comprises Methionin and arginine; Amino acid with uncharged polar head group of similar hydrophilicity value comprises following amino acid: leucine, Isoleucine, Xie Ansuan, glycine, L-Ala (analine), l-asparagine, glutamine, Serine, Threonine, phenylalanine, tyrosine.The Flk-1 that is equal on the function used herein is meant with VEGF or fragment and combines, but needn't have the acceptor of identical combination affinity of the natural Flk-1 of its correspondence.
Can design dna sequence dna of the present invention so that the Flk-1 encoding sequence becomes multiple end, this includes but not limited to the modification and the change of Expression of gene product.For example can use technology commonly known in the art, sudden change is introduced in for example site-directed mutagenesis, to insert new restriction site, changes glycosylation pattern phosphorylation etc.For example, in some expression system, as yeast, host cell may all make the gene product glycosylation.When using these expression systems, changing the Flk-1 encoding sequence may be preferable with the glycosylation site of eliminating any N connection.
In another embodiment of the invention, Flk-1 or modified Flk-1 sequence can cooperate so that fusion rotein is encoded with the stem sequence.For example, in order to screen the peptide storehouse, can be used for and to be expressed as the basic chimeric Flk-1 encoding histone of the anti-source decision of stem that commercial antibody is discerned.Also can design fusion rotein so that it contains the division site between Flk-1 sequence and stem protein sequence, thereby Flk-1 can be divided away from the stem part.
Can select in the embodiment at one of the present invention, can use the known chemistry of the prior art encoding sequence of synthetic Flk-1 whole or in part.For example referring to Caruthers.et al., 1980, Nuc.Acids Res.Symp.Ser.7: 215-233; Crea and Horn, 180, Nuc.Acids Res.9(10): 2331; Matteucci and Caruthers, 1980, Tetrahedron Letters 21: 719; And Chow and Kempe, 1981, Nuc.Acids Res.9(12): 2807-2817.On the other hand, this protein itself can use the synthetic whole or in part Flk-1 aminoacid sequence of chemical process.For example, peptide can be synthetic by solid phase technique, by discharging in the resin, and by preliminary high performance liquid chromatography purify (for example referring to Creighton, 1983, Proteins Structures And Molecular Principles, W.H.Freeman and Co., N.Y.pp.50-60).Can confirm composition (for example, the Edman degradation procedure of synthetic peptide by amino acid analysis or sequencing; Referring to Creighton, 1983, Proteins, Structures and Molecular Principles, W.H.Freeman and Co, N.Y., pp.34-49).2.Flk-1 the generation of the clone of receptor expression and expression Flk-1
In order to express bioactive Flk-1,, promptly contain in the carrier of transcribing and translate to neccessary composition to the insertion encoding sequence above described in 1. Flk-1 nucleotide sequence coding or function equivalent being inserted suitable expression vector.Can be used for multiple purpose with the Flk-1 gene product of recombinant expression vector or conversion and host cell or clone.These purposes comprise, but be not limited to, produce and this receptor bonded antibody (being mono-clonal or polyclonal), this comprises competitive inhibition in conjunction with VEGF and active those antibody of " neutralization " Flk-1, the VEGF analogue that screening and selection are worked by the Flk-1 acceptor or medicine or the like.
(1) expression system
The those of ordinary skill known method of prior art can be used to make up the expression vector that contains Flk-1 encoding sequence and suitable transcription/translation contrast signal.These methods comprise reorganization/gene recombination in stripped recombinant DNA technique, synthetic technology and the body.This class technology is as being found in Maniatis et al., 1989, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.and Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y..
Can use multiple host expresses carrier system to express the Flk-1 encoding sequence.These systems include, but are not limited to microorganism, as bacterium, the plasmid DNA that contains the Flk-1 encoding sequence or the cosmid DNA expression vector that transforms with the recombinant chou phage DNA; Insect cell system with the recombinant virus expression vector that contains the Flk-1 encoding sequence (for example baculovirus (baculovirrus)) infection; With the recombinant virus expression vector that contains the Flk-1 encoding sequence (for example, cauliflower mosaic virus, CaMV; Tobacco mosaic virus (TMV), TMV) infect or with the plasmid recombinant expression vector that contains the Flk-1 encoding sequence (for example, Ti-plasmids) plant transformed cell system; Or (for example with the recombinant virus expression vector, adenovirus, vaccinia virus) infected animals clone, comprise being designed in double small sheet karyomit(e), contain (CHO/ahfr) that amplifies that repeatedly duplicate or stable or the clone (for example mouse cell line) of the unstable Flk-1 DNA that amplifies.
The intensity and the specificity of the expression key element of these systems are variable.According to used host/vector system, in expression vector, can use any amount of suitable composition of transcribing and translate, this comprises constitutive character and inducible promoter.For example, when in the bacterium system, cloning, can use inducible promoter, as the PL of phage, plac, ptrp, ptac, (ptrp-lac hybrid promotor) etc.; When in insect cell system, cloning, can use such as promotors such as baculovirus, polyhedron promoters; When clone in the vegetable cell system, can use promotor (for example, the heat-shocked promotor that obtains by the vegetable cell genome; The little subunit of RUBISCO promotor; The conjugated protein promotor of chlorophyll a/b) or promotor (for example, the 35S RNA promotor of CaMV that obtains by plant virus; TMV coat protein promotor); When promotor that obtains at mammalian cell (for example, metallothioneins promotor) or promotor (for example, gland virus stage starting that obtains by mammalian virus; Vaccinia virus 7.5K promotor); When generation contains the clone of the Flk-1DNA that repeatedly duplicates, but can use SV40-, the BPV-with suitable selection marker and the carrier of EBV-base.
In the bacterium system, can preferentially select a series of expression vectors according to the purposes of the Flk-1 that is expressed.For example, when desire prepares lot of F lk-1 when producing antibody or screening peptide storehouse, it is desirable causing the carrier of the fusion protein product that highly expression is easy to purify.These carriers comprise, but be not limited to, intestinal bacteria (E.coli) expression vector PUR278(Ruther et al, 1983, EMBO is J.2: 1791) (wherein the Flk-1 encoding sequence can fit in this carrier in the framework with lac Z coding region, with the AS-lac Z albumen that hybridizes); PIN carrier (Inouye ， ﹠amp; Inouye 1985, Nucleic acids Res.13: 3101-3109; Van Heeke ﹠amp; Schuster, 1989, J.Biol, Chem, 264: 5503-5509); Or the like.Also can use the PGEX vector expression as xenogenesis polypeptide with fusion rotein of glutathione S-transferase (GST).Usually, these fusion roteins are soluble and can wash-out are easy to purify from the dissolved cell when having free glutathione to exist by being adsorbed on gsh-sepharose 4B subsequently.The PGEX carrier is designed to include zymoplasm or factor Xa proteolytic enzyme division site, so that significant polypeptide of being cloned can partly be discharged by GST.
In yeast, can use a series of carriers that contain composition or inducible promoters.In order to comment on, referring to Current Protocols in Molecular Biology, Vol.2,1988, Ed, Ausubel et al., Greene Publish.Assoc.﹠amp; Wiley Interscience, Ch.13; Grant et al., 1987, Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds, WU ﹠amp; Grossman, 1987, Acad, Press, N.Y., Vol, 153, PP.516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch.3; And Bitter, 1987, Heterotogous Gene Expression in yeast, Methods in.Enzymology, Eds.Berger ﹠amp; Kimmel, Acad.Press, N.Y., Vol.152, PP.673-684; And TheMolecular.Biology of the Yeast Saccharomyces, 1982, Eds.Strathern et al., Cold Spring Harbor Press, Vols, I and II.
Under the occasion of using plant expression vector, the expression of Flk-1 encoding sequence can be caused by any promotor.For example, can use viral promotors, as 35SRNA and 19SRNA promotor (the Brisson et al. of CaMV, 1984, Nature 310: 511-514), or the coat protein promotor of TMV (Takamatsu et al., 1987, EMBO J.6: 307-311); On the other hand, can use plant promoter, as the little subunit of RUBISCO (Coruzzi et al., 1984, EMBO J.3: 1671-1680; Broglie et al., 1984, Science 224: 838-843); Or the heat-shocked promotor, for example soybean hsp17.5-E or hsp17.3-B(Gurley et al., 1986, Mol.Cell.Biol, 6: 559-565).Can utilize Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electroporation etc. with in these member introduced plant cells.In order to comment these technology, for example referring to Weissbach ﹠amp; Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section Vlll, pp.421-463; And Gyierson ﹠amp; Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch.7-9.
The selective expression system that can be used for expressing Flk-1 is the insect system.In a kind of such system, use Autographa Californica nuclear polyhidrosis virus (AcNPV) as expressing heterogeneic carrier.This virus is grown in Spodoptera frugiperda cell.The Flk-1 encoding sequence can be cloned into the non-intrinsically safe district (for example polyhedron gene) of virus and place a kind of AcNPV promotor (for example polyhedron promoter) control down.The smooth insertion of Flk-1 encoding sequence can cause the inactivation of polyhedron gene and produce unsealed recombinant virus (promptly lacking by the virus of polyhedron gene encoded protein matter shell).Utilize these recombinant virus to infect Spodoptera frugiperda cell then, the gene of this insertion has therein been expressed (for example referring to Smith et al., 1983, J.Viol.46: 584; Smith, U.S.Patent NO.4,215,051).
In mammalian host cell, can use the expression system of a series of virus bases.Using under the occasion of adenovirus as expression vector, the Flk-1 encoding sequence can be connected on the adenovirus transcription/translation contrast complex body, for example late promoter and three fens leader sequences.This mosaic gene can be inserted in the adenoviral gene group by recombinating in stripped or the body then.Insert recombinant virus that virus genomic non-intrinsically safe district (for example E1 and E3 district) meeting formation is lived and expressed Flk-1 in the host who infects (for example, referring to Logan ﹠amp; Shenk, 1984, Proc, Natl, Acad.Sci.(USA) 81: 3655-3659 can use cowpox 7.5K promotor (for example referring to Mackett et al., 1982, Proc.Natl.Acad.Sci.(USA) 79: 7415-7419 on the other hand; Mackett et al., 1984, J.Virol.49: 857-864; Panicali et al., 1982, Proc.Natl.Acad.Sci.79: 4927-4931).
Also may need specific start signal to translate with the Flk-1 encoding sequence that inserts effectively.These signals comprise ATG initiator codon and flanking sequence.At complete Flk-1 gene, comprise that its oneself initiator codon and flanking sequence are inserted under the occasion in the suitable expression vector, then without any need for the additional contrast signal of translating.Yet, under the occasion that only some Flk-1 encoding sequence is inserted into, must provide the external source that comprises the ATG initiator codon to translate contrast signal.In addition, this initiator codon must be read a yard homophase with the opening of Flk-1 encoding sequence, to guarantee translating of all insets.These external sources are translated contrast signal and played a codon can be multiple source, natural and synthetic.The efficient of expressing can by comprise suitable transcribe enhancement factor composition, transcription termination region etc. be enhanced (referring to Bittner et al., 1987, Methods in Enzymol.153: 516-544).
In addition, required mode that can be specific selects to regulate the expression of insertion sequence or the host cell strain of modification and processing gene product.This class of protein product is modified (for example glycosylation) and handled (for example division) may be important to proteic function.Different host cells to proteic translate aftertreatment and modify have distinctive and special mechanism.Can select suitable clone or host system to guarantee to be expressed the correct modification and the processing of foreign protein.For this reason, can use the eukaryotic host cell of the cell mechanism that the primary transcription, glycosylation and the phosphorylation that have gene product suitably handle.These mammalian host cells include, but not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 2P3, WI38 or the like.
For long-term, high yield produces recombinant protein, preferably stably express.For example, but design stability is expressed the clone of Flk-1.(for example would rather use by suitable expression control composition, promotor, enhancement factor sequence, transcription termination region, polyadenylation site etc.) the Flk-1 DNA that controlled, with the selective marker transformed host cell, and do not use the expression vector that contains the virus replication source.After introducing different DNA, designed cell was grown 1-2 days in the substratum of enriching, be converted in the selective medium then.Selective marker in the plasmid recombinant has non preference can and can make cytotostatic ground go in its karyomit(e) plasmid integration and the focus of growing and can be cloned into and be extended to clone successively to form.This method can be advantageously used in design at this cell surface expression Flk-1, and replys the clone of the signal transduction of VEGF transmission.She Ji clone is particularly useful to screening VEGF analogue like this.
Can use a series of selective systems, these systems include, but are not limited to respectively can be at tk -, hgprt -Or aprt -The herpes single type virus thymidine kinase that uses in the cell (Wigler, et al., 1977, Cell 11: 223), hypoxanthine guanine phosphoribosyltransferase (Szybalska ﹠amp; SzYbalaki, 1962, Proc.Natl.Acad.Sci.USA 48: 2026), and the gene of adenine phosphoribosyl transferase (Lowy, et al., 1980, cell 22: 817).Also have, the resistance that can utilize metabolic antagonist is as selecting dhfr(Wigler with ammonia methylpurine resistance, et al., 1980, Natl.Acad.Sci.USA 77: 3567; O ' Hare, et al., 1981, Proc.Natl.Acad.Sci USA 78: 1527); Gpt(Mulligan ﹠amp with mycophenolic acid resistance; Berg, 1981, Proc.Natl.Acad.Sci.USA 78: 2072); Neo(Colberre-Garapin with aminoglycoside G-418 resistance, et al., 1981, J.Mol.Biol.150: 1); With the hygro Santerre with hygromycin resistance, et al., 1984, Gene 30: 147) basis of gene.Recently, disclose other selection gene, as made cell utilize indoles to replace the trpB of tryptophane; Can make cell utilize histidinol (histinol) to replace the his D(Hartman ﹠amp of Histidine; Mulligan, 1988, Proc.Natl.Acad.Sci.USA 85: 8047; ) and have anti-2,5-diaminovaleric acid Guang carboxylic acid inhibition, 2-(difluoromethyl)-the ODC(2,5-diaminovaleric acid Guang carboxylic acid of DL-2,5-diaminovaleric acid, DFMO resistance) (Meconlogue is L.1987, In: Current Communications in Molecular Biolgy, Cold Spring Harbor Laboratory ed.).
(2) express the transfectant of Flk-1 or the discriminating of transformant
At least can differentiate the host cell of the gene product that contains encoding sequence and watchcase physiologically active by four kinds of common methods; (a) DNA-DNA or DNA-RNA hybridization; (b) exist or do not have " mark " gene function; (c) evaluation is detected by immunoassay or by the measured gene product of its biological activity by the measured transcriptional level (d) of Flk-1mRNA transcript in the expression host cell.
In first method, can contain respectively by use and hybridize the existence that detects the Flk-1 encoding sequence that in expression vector, is inserted with the DNA-DNA or the DNA-RNA of Flk-1 encoding sequence or its albumen or derivatives thereof homologous nucleotide sequence probe.
In the second approach, can according to be with or without some " mark " gene function (for example, thymidine kinase activity, to antibiotic resistance, to the resistance of methotrexate, transform phenotype, the formation or the like of occlusion body in baculovirus) differentiate and select recombinant expression vector/host system.For example, if the Flk-1 encoding sequence is inserted in the marker gene sequence scope of carrier, then can not differentiate the recombinant chou that contains the Flk-1 encoding sequence by not having marker gene function.On the other hand, marker gene can be placed on and the expression of the placed in-line position of Flk-1 sequence with control Flk-1 encoding sequence under the identical or different promotor control of usefulness.According to inducing or selecting, the expression of mark has shown the expression of Flk-1 encoding sequence.
In the third method, can measure the transcriptional activity of Flk-1 coding region by cross experiment.For example, can separate with the Northern trace of Flk-1 encoding sequence or its specific position homologous probe and analyze RNA by using.Be these probe hybridizations, can extract and test the total nucleic acid of host cell.
In the 4th kind of method, for example can pass through Western trace, immunoassay, connect the Flk-1 protein product is estimated in immunoassay or the like from immunology expression as radioimmunoprecipitation, enzyme.Yet, the final test of the success of this expression system is related to the active Flk-1 gene product of detection of biological.Can use multiple test to detect receptor active, the clone that includes, but not limited to use design as the VEGF of test matrix in conjunction with mensuration; The VEGF biological assay.
3.Flk-1 the purposes of the clone of acceptor and design
Vascularization, new capillary vessel growth needs a series of physiological processs, and its scope is grown until placenta from wound healing, tissue and neomorph, gestation back placentation.The paraplasm of blood vessel is multiple disease, as rheumatic arthritis, retinopathy and psoriasic important factor.Vascularization also is to depend on angiopoietic solid tumor growth and multiple for active important factor.Thereby, can use on the medicine angiopoietic inhibition with treatment because or the malignant tumour of following disease that aberrant angiogenesis growth causes and treatment to relate to the growth and the diffusion of solid tumor.
In one embodiment of the invention, can use the clone Flk-1 acceptor and/or that express the Flk-1 acceptor to screen vascularization or the antagonist of vasculation or antibody, peptide or other part of antagonist that transmits as by the Flk-1 acceptor.For example, can use the active anti-Flk-1 antibody of energy neutralize VEGF to suppress the Flk-1 function.In addition, can select to be used for wound healing to the very similar anti-Flk-1 antibody of VEGF activity.On the other hand, the solubility Flk-1 albumen of expressing with recombinant chou or express the proteic cell line selection peptide of Flk-1 storehouse for having differentiated that by the biological activity that suppresses Flk-1 the treatment molecule of the sort of effect is effective.
In one embodiment of the invention, can use cell line selection and the discriminating VEGF antagonist and the antagonist of the design of The expressed Flk-1 coding region or its ligand binding region.Available serial of methods screening synthetic compound, natural product and other potential biological active materials source.Can use as embodiment 1.(9 hereinafter) described in those standard receptors bind technical measurement test compounds to suppressing VEGF and Flk-1 bonded ability.The ability of reagent prevention or imitation, VEGF can be determined in conjunction with the effect that signal transduction is reacted on the F1k-1 express cell.For example, can monitor such as the F1k-1 kinase activity activate, secondary messenger in the cellular metabolism produces or this class of adjusting of changing is replied.These mensuration can be utilized as these purposes and the routine techniques developed carries out.
(1) with the cell line selection peptide storehouse of Flk-1 albumen or design
Can use by be connected amino acid on the solid phase carrier all may make up the random peptide library of being formed differentiate can with the given acceptor or the functional area of other acceptor, ligand-binding site point bonded peptide (Lam as the kinases district, K.S.et al., 1991, Nature 354: 82-84).Screening peptide storehouse may find to play the value that has aspect the medicament of inhibition receptor biological active function in the medical treatment by them and given acceptor interaction.
Discriminating can be by finishing with recombinant chou solubility Flk-1 protein screening peptide storehouse with Flk-1 bonded molecule.At 2.(1 above) in the method for expression and purification Flk-1 has been described, this method can be used for the fragment of expression recombinant total length Flk-1 or Flk-1 according to interested functional area.For example, the outer ligand binding domain of the kinases of Flk-1 and born of the same parents can be expressed separately and is used to screen the peptide storehouse.
In order to differentiate and to separate the peptide/solid phase carrier that interacts and form the complex body that contains Flk-1, must spike or " mark " Flk-1 molecule.Can be with Flk-1 albumen and enzyme, as alkaline phosphatase or horseradish peroxidase or with other reagent, be connected as fluorescent marker, these markers can comprise fluorescein isothiocyanate (FITC), phycoerythrin (PE) or rhodamine.Can utilize routine techniques in the prior art to carry out being connected of any given marker and Flk-1.On the other hand, the Flk-1 expression vector can be designed to express to contain and to buy the chimeric Flk-1 albumen that antibody is the epitope that exists market.Can use this epitope specific antibodies of method mark commonly known in the art, this comprises the pearl mark with enzyme, fluorescence dye or painted or magnetic.
With random peptide library " mark " Flk-1 combination is cultivated 30 minutes to 1 hour so that form complex body between the peptide kind at Flk-1 and Ku Nei at 22 ℃.Then this storehouse is washed to remove any unconjugated Flk-1 albumen.If Flk-1 combines with alkaline phosphatase or horseradish peroxidase, the matrix that then impouring of whole storehouse is contained alkaline phosphatase or peroxidase, for example 5-bromo-4-chloro-3-indyl (indoyl)-phosphoric acid ester or 33 ', 4,4 " in diaminobenzidine (diamnobenzidine) culture dish (DAB).After cultivating several minutes, peptide/solid phase-Flk-1 complex body changes color, and can be easy to be differentiated and having physical sepn under the dissecting microscope of micromanipulator.If used fluorescently-labeled Flk-1 molecule, then can separate this complex body by the fluorescence-activation classification.If used the chimeric Flk-1 albumen of expressing heterologous epitope, then can finish the detection of peptide/Flk-1 complex body by the epitope specific antibodies of applying marking.In case after separating, can measure the identity that is connected the peptide on the solid phase carrier by sequencing peptides.
Except using solubility Flk-1 molecule, in another embodiment, can use whole cell to detect and cell surface receptor bonded peptide.For with many subunits and unsettled acceptor or for the application of the acceptor in the lipid district of the cytolemma that plays function at needs, preferably use intact cell.At 2.(1 above) and 2.(2) in the method that produces the clone of expressing Flk-1 has been described.Used cell had both been lived in this technology also fixed cell.These cells will with random peptide library cultivate and will with some peptide in this storehouse in conjunction with between target cell and relevant solid phase carrier/peptide, to form " rosette ".This rosette can or be removed with physical method under dissecting microscope by the differential centrifugation separation then.
As the alternative method of complete raji cell assay Raji, with regard to the acceptor in cytolemma lipid district that membrane-bound acceptor has maybe needed function, acceptor molecule can be reconstructed into the liposome that can connect spike or " mark ".
(2) antibody produces and screening
The whole bag of tricks well known in the prior art all can be used for producing the antibody of the epitope that resists the Flk-1 acceptor that produces through reorganization.The fragment that these antibody include, but are not limited to polyclone, mono-clonal, chimeric, strand, Fab fragment and produced by a Fab expression library.For example, those neutralizing antibodies of contention acceptor VEGF binding site to diagnosis and treatment specially for preferably.
Can will carry out radio-labeling so that traceable its position and the distribution after injecting in vivo of people in conjunction with the monoclonal antibody of Flk-1.Can use radiolabeled antibody as the refigure angiopoietic non-invasive diagnosis instrument relevant with a series of diseases, these diseases comprise the formation and the transfer of rheumatic arthritis, retinal degeneration, tumour.
Also the immunotoxin at cytotoxic agent can be located at intravital specific position.For example, can be with the specific monoclonal antibody of high-affinity and bacterium or plant poison, as diptheria toxin, abrin or the complexing of ricin covalency.The universal method of preparation antibody/hybrid molecule can comprise uses the sulfydryl linking agent, as SPDP, this linking agent with main amine groups attached on the antibody and by disulfide exchange with toxin attached on the antibody.Can use hybrid antibody to eliminate the Flk-1 that expresses endotheliocyte specially.
In order to produce antibody, can make various host animals make immunization by injection Flk-1 albumen, these animals include, but are not limited to rabbit, mouse, rat or the like.Can use various adjuvants to reply according to host type with enhancing immunity, these adjuvants comprise, but be not limited to Freund ' s(fully and incomplete), inorganic gel, as aluminium hydroxide, surfactant, cry out hemocyanin, dinitrophenol(DNP) and the potential adjuvant useful as lysolecithin, epoxy ethane-epoxy propane polyvalent alcohol, polyanion, peptide, oil-emulsion, key hole, as the BCG(bacille Calmette-Guerin vaccine to human body) and Corynebaterium parvum.
Anti-Flk-1 monoclonal antibody can provide any technology preparation that produces antibody molecule by using by passage cell in the culture.These technology include, but are not limited to by the disclosed the earliest hybrid knurl of Kohler and Milstein (hybridoma) technology (Nature, 1975,256: 495-497), human body cell hybrid knurl technology (Kosbor et al., 1983, Immunology Today, 4: 72; Cote et al., 1983, Proc.Natl.Acad.Sci., 80: 2026-2030) with EBV hybrid knurl technology (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp.77-96).In addition, can use by being connected technology (the Morrison et al. that generation " chimeric antibody " is developed with gene from the gene of the mouse antibodies molecule of suitable antigen-specific from suitable bioactive human body antibody molecule, 1984, Proc.Natl.Acad.Sci., 81: 6851-6855; Neuberger et al., Nature, 312: 604-608; Takeda et al., 1985, Nature, 314: 452-454).On the other hand, can be suitable for producing the specific single-chain antibody of Flk-1 for producing the described technology of single-chain antibody (United States Patent (USP) 4,946,778).
Can produce the antibody fragment that contains specific Flk-1 binding site by known technology.For example, these fragments include, but are not limited to: the F(ab ' that can produce by the gastric pepsin digestion of antibody molecule) 2Fragment and can be by reducing F(ab ') 2Segmental disulphide bridges and the Fab fragment that produces.On the other hand, can make up the Fab expression library to differentiate having required specific mono-clonal Fab fragment fast and easily to Flk-1.
4.Flk-1 the purposes of encoding sequence
The Flk-1 encoding sequence can be used to detect the diagnostic purpose that Flk-1 expresses.What comprise in scope of the present invention is the oligonucleotide sequence, and they comprise sense-rna and dna molecular and the ribozyme that suppresses the FIK-1 functional transcription is arranged.In addition, the mutant that can express the Flk-1 with the negative effect of dominance in the target cell population is with the activity of the wild-type Flk-1 that suppresses endogenous expression.
(1) purposes of Flk-1 encoding sequence in diagnosis and treatment
Flk-1 DNA may have some purposes to diagnosing the disease that causes owing to the Flk-1 unconventionality expression.For example, the Flk-1 dna sequence dna can be used in vivisection or the Autopsied hybridization assays unusual with what diagnose Flk-1 to express; For example, Southern or Northern analyze, and this comprises in situ hybridization mensuration.
Flk-1 DNA can be used as the expression of probe in detecting Flk-1 mRNA.In a special embodiment as herein described, the Flk-1mRNA that has analyzed in the different developmental phases mice embryonic expresses.The Northern engram analysis has shown that a large amount of 5.5kb mRNA volumes are expressed between 9.5 days and 18.5 days, and finishes obviously decline (Fig. 2 A) near gestation.After birth, in 4-8 days brain kapillaries, compare, find Flk-1 mRNA highly enriched (Fig. 2 B), hinted that Flk-1 is to the outgrowth effect of endotheliocyte with total cerebral RNA.
For the more detailed information that obtains expressing about Flk-1 during fetal development and in the commitment of vascular development, by following embodiment part 1.(4) described in, carried out the in situ hybridization test, in situ hybridization has confirmed that the expression of Flk-1 in the mouse embryo growth period mesosome is limited to endotheliocyte and precursor (Fig. 3 and Fig. 4) thereof basically.As (1980) as described in the Bar, endotheli ocytosis that Flk-1 is found in embryo and brain and time and space expression pattern and neural growth are closely related be the physiological process of feature during, in endotheliocyte, expressed.The vascular bud that in enclosing neuroplexus, produces radially grow into neural outer and from then on branch and also find that these buds express lot of F lk-1 mRNA(Fig. 5).Commitment after birth, endotheli ocytosis still clearly and Flk-1 expressed, and in the adult organism body, after vascularization is finished, the reduction that the decline of endotheli ocytosis is expressed corresponding to Flk-1.
The oligonucleotide sequence that has comprised the sense-rna that suppresses the Flk-1mRNA translation and dna molecular and ribozyme also within the scope of the invention.Sense-rna and dna molecular are by combining and prevent that with target mRNA albumen from translating directly works to block mRNA and translate.With regard to antisense DNA, preferably by the rotaring intertranslating start site, for example-10 of the Flk-1 nucleotide sequence and+the low deoxynucleotide that obtains between 10 districts.
Ribozyme is can the special splitted ribozyme of catalysis RNA molecule.The mechanism of ribozyme effect comprises the sequence specific cross of nuclear 1 enzyme molecule and additional target RNA, dissolving division in the nucleus subsequently.Ribozyme molecule design specific and the interior dissolving of catalysis Flk-RNA sequence nucleus splitted hammerhead shape characteristic effectively within the scope of the present invention.
The observation target molecule is passed through in special nucleus division site in any potential RNA target at first, comprises following sequence, and nuclear 1 enzyme of GUA, GUU and GUC divides the site and obtains discriminating.In case after being differentiated, can estimate expected structure characteristic, as making nucleotide sequence unfavorable secondary structure that becomes corresponding to short rna sequence between 15 and 20 Nucleotide that contain this target gene district, site.The suitability of candidate target also can obtain evaluation by using ribonuclease protection assay to detect its accessibility with the hybridization of additional oligonucleotide.
Sense-rna of the present invention and dna molecular and ribozyme all can be by the method preparations of synthetic RNA molecule well known in the prior art.These methods comprise the technology of the low deoxynucleotide of chemosynthesis commonly known in the art, for example solid phase phosphoramidate chemosynthesis.On the other hand, the RNA molecule can produce antisense rna molecule coded DNA sequence by transcribing in stripped and the body.These dna sequence dnas also can be impregnated in and be combined with suitable R NA polymerase promoter, as the kind of T7 or SP6 polymerase promoter carrier widely.On the other hand, depend on the structure of used promotor or generally the Antisense cDNA member of synthesize antisense rna can introduce clone with being stabilized.
Can introduce various modifications to dna molecular as the means that improve stability and half-life.Possible modification includes, but are not limited to the flanking sequence of Nucleotide or deoxynucleotide is added 5 of molecule ' and/or 3 ' terminal or uses thiophosphatephosphorothioate or 2 ' 0-methyl rather than low deoxynucleotide master intrachain phosphodiesterase key.
(2) purposes of the negative Flk-1 mutant of dominance in gene therapy
Turn into being considered to provide a kind of by part inductive receptor dimerization the coupling part is played allosteric conditioning signal in conjunction with the kinase activity hormesis.The defective acceptor can play the negative mutation effect of dominance by suppressed normal receptor activation and response by forming of unfruitful heterodimer.Thereby the defective acceptor can be designed to the recombinant virus carrier and be used for expressing inadequately the gene therapy of the individuality of Flk-1.
In one embodiment of the invention, can be by in selecteed cell, expressing the Flk-1 molecule of differentiating mutant forms with the negative effect of dominance.Keep formation to have wild-type Flk-1 albumen, but the Flk-1 of the ability of the dipolymer that can not work in signal transduction disappearance or missense mutant can be used for suppressing the biological activity of endogenous wild-type Flk-1.For example, the cell kinase district of Flk-1 can lack, and the result forms the dimerization effect still can stand with endogenous wild-type receptor, but truncate Flk-1 molecule that can not change over signal.
The paraplasm of blood vessel is multiple pathogenic disorder, as rheumatic arthritis, retinopathy and a psoriasic important factor.Uncontrolled vascularization also is the growth of solid tumor and an important factor in the transfer.Recombinant virus can be designed to express the Flk-1 that can be used for suppressing the negative form of the active dominance of the endogenous Flk-1 of wild-type.These virus can be used for the treatment of in the medical treatment by Flk-1 unconventionality expression or the active disease that is caused.
By virus, as retrovirus, vaccinia virus, adeno-associated virus, simplexvirus, or the expression vector that obtains of bovine papilloma virus, can be used for recombinant chou Flk-1 is passed in the target cell population.Can use those of ordinary skill known method in the prior art to make up the recombinant virus carrier that contains the Flk-1 encoding sequence.For example be illustrated in " Mamiatis et al.; 1989; Molecular Cloning A Laboratory Manual; Cold Spring Harbor Labratory; N.Y.and Ausubel et al., 1989, Current Protocols in Molecular Biology; Grene Publishing Associates and Wiley Interscience, the technology described in the N.Y..On the other hand, recombinant chou F1k-1 molecule can be rebuild or is sent to the liposome of target cell.
In a particular of the present invention, the deletion mutant of Flk-1 acceptor is designed to recombinant chou and transcription vector.Two kinds of clone and separate things that are called as pLXSN Flk-1 TMcl.1 and pLXSN Flk-1 TMcl.3 contain the truncate Flk-1 acceptor that lacks the 561COOH terminal amino acid.In order to obtain producing the virus of clone,, use the conditioned medium that contains virus to infect the GPE cell subsequently with recombinant vector transfection PA37 cell.
For whether the expression of the mutant of test signal defective suppresses endogenous Flk-1 receptor active, with C6 rat glioblastoma cell (tumour cell) with the mouse cell that produces retrovirus mixes and subcutaneous injection is gone in the nude mice.Usually, tumor cell injection is gone into nude mice and can be caused the hyperplasia of tumour cell and the vascularization of formed lump.So the essential reason that is considered to form blood vessel owing to Flk-1 may work the vascularization that suppresses developmental tumour by the blocking-up Flk-1 activity of truncate expression of receptor, suppresses its growth whereby.Illustrated as Figure 13 and 14, that works in coordination with injection generation cell virus compares remarkable inhibition growth of tumor to express truncate Flk-1 acceptor with the comparison of only accepting tumour cell.
5.Flk-1 the purposes of acceptor or part
The interaction of receptor/ligand is considered to play an important role in the signalling system during vasculation and vascularization between Flk-1 and the VEGF.The paraplasm of blood vessel is the important factor of a series of diseases.
The expression of Flk-1 RNA and the growth of brain and relevant with endotheli ocytosis, this has represented that Flk-1 may be the relevant acceptor of RST adjustment a kind of and in the vascularization process.VEGF shown be a kind of known act on specially mitotic growth factor on the endotheliocyte (Ferrara, N.and Henael, W.J., 1989, Biochem.Biophys.Res.Comm 161: 851-858).By embodiment 1.(9 hereinafter) and 1.(10) described in carried out crosslinked and part in conjunction with test to determine that whether VEGS is the part of a kind of Flk-1 and the result shows, Flk-1 is a kind of reliable high-affinity vegf receptor (Fig. 9).
In one embodiment of the invention, can be with Flk-1 part, Flk-1 acceptor itself, or contain in the sheet segment body of its VEGF binding site and use to regulate vascularization and/or vasculation.For example, thus use the Flk-1 acceptor or contain the fragment of this VEGF binding site can be competitively with VEGF combine and in vivo inhibition its with the interaction inhibition vascularization and/or the vasculation of natural Flk-1 acceptor.On the other hand, the part of Flk-1 comprises that anti-Flk-1 antibody or its fragment can be used for regulating vascularization and/or vasculation.The active antagonist of VEGF can be used for promoting wound healing.And the active antagonist of VEGF can be used for suppressing tumor growth.
Can prepare these medicaments and make system or topical application according to the particular case of being treated.Can be at " Remington ' s Pharmaceutical Sciences, " (Mack Publishing Co., Easton, PA) technology that can find preparation and use in last edition.That suitable method can comprise is oral, rectum, stride dispenser in mucous membrane or the intestines; Parental generation transmits, comprise in muscle, subcutaneous, intramedullary injection and the film, directly in the ventricle, in the vein, intraperitoneal, nose, or intraocular injection, more than only listed seldom several.In order to inject, medicament of the present invention can be mixed with aqueous solution, preferably in the buffer reagent of physical compatibility, and as Hanks solution, Ringer solution, or physiological saline buffer.For striding the mucous membrane dispenser, in prescription, can use the permeate agent that is suitable for and does not go into obstacle.These permeate agents are known usually in the prior art.
Embodiment: the clone and the expression pattern of Flk-1 high-affinity vegf receptor,
With clone and the feature that the lower section is described and Flk-1cDNA clones.Northern trace and in situ hybridization analysis revealed Flk-1 are expressed in endotheliocyte.Crosslinked and part has pointed out further that in conjunction with test Flk-1 is a kind of high-affinity vegf receptor.
1. material and method
(1) cDNA of Flk-1 clone
The DNA(Fahrner et al. that extracts in the λ gt10 cDNA storehouse of use by 8.5 days mice embryonics, 1987, EMBO.J.6: 1497-1508) as the template (PCR of polymerase chain reaction; Saiki, R.K.et al., 1985 Science 230: 1350-1354).At one independently in the method, to be used for amplification (Risau by the cDNA of isolating capillary endothelial cells in the 4-8 days mouse brains in birth back, W., 1990 In: development of the Vascular System.Issues Biomed.Basel Karger 58-68 and Schnurch et al., do not publish).According to the primer (Wilks, A.F., 1989.Proc, the Natl.Acad.Sci.U.S.A.86: 1603-1607) that design degeneration in amino acid identity by the total kinases district inner height of whole RTK.
Use 32(the Feinberg of P-mark, A.P.and Vogelstein, B.1983 Anal.Biochem.132: 6-13) 210-bp PCR fragment with the cDNA clone of total length Flk-1 with by according to OKayama and Berg(1983) another 8.5 days mice embryonic brain cDNA storehouses of making of method, and 11.5 days mice embryonic λ gt11(Clonetech) separate.
(2) mice embryonic
The mating of Balb/c mouse spent the night and morning vagina detect and to be confirmed as gestation 1/2 day.With 5M guanidine radicals thiocyanic ester with frozen embryo homogenize with carry out the Northern engram analysis and by (Ullrich, A.et al., 1985, Nature 313: 756-761) described isolation of RNA.And be kept at-70 ℃ before use, this embryo is at Tissue-TeK(Miles) in embedding, at the liquid nitrogen surface freezing to carry out in situ hybridization.
(3) preparation of probe
2619bp on 5 ' position of receptor cdna (Promega) in pGem 3 district's carriers is cloned as the EcoR1/BamH1 fragment.By use by Hexanucleotide at random cause α- 32PdATP(Amersham) mark cDNA fragment makes the probe (Boehringer of Northern blot hybridization; Feinberg, A.P.and Vogelstein, B., 1983 Anal.Biochem.132: 6-13).
For carrying out in situ hybridization, press Schnuch and Risau(Development, 1,991 111: 1143-54) described preparation strand antisense DNA probe.Make the plasmid linearize and use the SP6RNA polysaccharase to synthesize meaningful transcript (Boehringer) at 3 of cDNA ' end.Use DNA enzyme liberating DNA(RNAase free preparation, Boehriger Mannheim).By with the MMLV reversed transcriptive enzyme (BRL) reverse transcription, with this transcript carry out α- 35S dATP(Amersham) synthetic with the cDNA that causes at random.For the little cDNA fragment of average about 100bp of obtaining being suitable in situ hybridization, use big excessive primer.Subsequently in 100mMNaOH in 70 ℃ with rna transcription this part hybridization 20 minutes, and with the HC1 of same amount this probe that neutralizes, with the purification of Sephadex C50 post.Behind ethanol sedimentation, be 5 * 10 with final activity 5Cpm is with this probe dissolving.Prepare meaningful probe to compare hybridization with same procedure.
(4) RNA extracts and the Northern analysis
According to Chromczynski and Sacchi(1987) sour phenol method separate whole kytoplasm RNA.1.2% agarose formaldehyde (Sambrook, J.et al., 1989 Molecular Cloning: A Laboratory Manual 2nd ed.Cold Spring Harbor Laboratory Press) in the gel with Poly(A +) RNA aliquots containig electrophoresis and be transferred to nitrocellulose membrane (Schleicher ﹠amp; Schuell) on, at 50% methane amide, 5 * SSC(750mM sodium-chlor, 75mM Trisodium Citrate) 5 * Denhardt ' s(0.1%Ficoll 400,0.1% polyvinylpyrrolidone (Polyvinylpryollidone), 0.1%BSA) and 0.5%SDS in 42 ℃ with 1-3 * 10 6Cpm-ml -1 32The dna probe that p-causes is at random hybridized, and washs in 50 ℃ of high strength in 0.2 * SSC, 0.5%SDS subsequently.Filtrate is exposed 4-8 days.
(5) in situ hybridization
Basically carry out the back fixing and hybridization of subclone according to people's such as Hogan (1986) method.In the Leitz thermostat container, cut 10 μ m slabs in-18 ℃.Prehybridization is handled the proteic substantially cultivation of removal of not using 0.2M HCl to carry out.Containing 50% methane amide, 300mM NuCl, 10mMTris-HCl, 10mM NaPo 4(pH 6.8), 5mM EDTA, 0.02%Ficoll400,0.01% polyvinylpyrrolidone (polvinylprolidone), 0.02%BSA, 10m/ml yeast rna, 10% asuro and 10mM NaCl, 10mM Tris-Hcl, 10mM NaPo 4(PH6.8), 5mM EDTA, in the buffer reagent of 10mM DTT in 52 ℃ with 35The S-cDNA probe is cultivated the part of these cuttings.Apply wave carrier piece and exposure 8 days to carry out radioautogram with Kodak NTB2 film emulsion.After the development, these sections are redyed with toluidine blue or May-Grinwald.
(6) sero-fast preparation
To comprise Flk-1 the 128c end amino acid 3 ' the EcoRV/Hind II fragment subclone (Smith, D.B.and Johnson, K.S., 1990 Gene.67: 31-40 in fusion protein expression vector pGEx3x that cause; Phamacia).By purify this fusion rotein and be used to make the rabbit immunity of described method.As for making the used and described method of rabbit immunity this fusion rotein of purifying.After secondary strengthens, be used for immunoprecipitation with the rabbit blood drawing and with antiserum(antisera).
(7) transition of Flk-1 in the COS-1 cell expressed
Basically by Chen and Okayama(1987 Mol.Cell.Biol.7: 2745-2752) and people such as Gorman (1989 Virology 171: 377-385) described method is carried out the transfection of COS-1 cell.Putting it briefly, is 1.0 * 10 with these cell inoculations to density 6/ 10-cm ware is also cultivated in the DMEM that contains 10% tire calf serum (Gibco) and is spent the night.20 μ g are cloned into the 0.25MCaCa that receptor cdna in the expression vector of cytomegalovirus promoters driven is blended in 0.5ml 2, 0.5ml 2 * BBS(280mMNaCl, 1.5mM Na 3HPO 4, 50mMBES, pH6.96) in and cultivated 30 minutes in room temperature.Then this calcium phosphate/DNA solution is added in this cell, slightly reverberate, and at 3%CO 2Cultivated 18 hours in 37 ℃ down.With cell by isolating on the flat board and handle as follows to carry out part in conjunction with test.
For obtaining the VEGF conditioned medium, with cell transfecting in the 15cm culture dish.Collect substratum after 48 hours also by the affinity chromatograph analysis part purification VEGF of use heparin High Trap TM post (pharmacia) and by ultrafiltration centrifugation (Ferrara, N.and Henzel, W.J.1989 Biochem.Biophys.Res.Comm.161: 851-858) make it concentrated.Measure the concentration of VEGF by the part competition experiments of carrying out with bovine aortic endothelial cells.
Measure in order to carry out autophosphorylation, with cell inoculation at 6 well culture dish (2 * 10 5Cell/every well), carries out transfection as described above, and in the DMEM that contains 0.5% tire calf serum, make it hungry 24 hours.These cells were handled 10 minutes or stayed with 500 PMVEGF at 37 ℃ then and do not handle then with the described method dissolving of people such as Kvis (1985).With the antiserum(antisera) of the anti-acceptor end that obtains in the rabbit with the Flk-1 immunoprecipitation.On the 7.5%SDS polyacrylamide gel, separate this immunoprecipitate, be transferred on the nitrocotton, and use at the mouse monoclonal antibody of phosphorus tyrosine and cultivate (5E2; Fendly, B.M.et al., 1990 Cancer Research 50: 1550-1558).Use the goat anti-mouse antibody and the ECL of horseradish peroxidase coupling Cm(Amersham) detection system observation protein band.
(8) radioiodination of VEGF
With recombinant chou human body VEGF(5 μ g: provide by Dr, H, Weich generosity) be dissolved in the sodium phosphate buffer of 110 μ l pH7.6 and with Hunter and Greenwood(1962) the method iodate.With reaction product by separating with this labelled protein, and before with 20% trichloroacetic acid precipitation and afterwards the aliquots containig of collected cut is counted with the cross-linked glucose G50 post of sodium phosphate buffer salt solution (PBS) pre-equilibration that contains 0.7% bovine serum albumin (BSA).As being measured by gel electrophoresis, this iodate degree of purity of production is calculated as and surpasses 90%, and specific activity is 77000cpm/ng.By using by Clauss, M.et al., (1990 J.Exp.Med.172: 1535-1545) described tissue factor introduce measure by with the biological activity of natural VE GF relatively, confirm the biological activity of iodate VEGF.
(9) VEGF and Flk-1's is crosslinked
Use 200PM at 4 ℃ 125I-VEGF will temporarily express the COS-1 cell of Flk-1 and the COS-1 cell culture of untransfected is spent the night, and be that with PBS washing secondary and 4 ℃ of exposures the 0.5mM two succinimide based substrates (DSS) of PBS were through 1 hour then.With these cytolysis, the Flk-1 immunoprecipitation, and by electrophoresis on 7% polyacrylamide gel, radioautogram analysis subsequently.
(10) VEGF combination
By top described (Schumacher, R.et al., 1991, J.Biol.Chem.266: method 19288-19295) has been carried out part in conjunction with test, will be among the DMEM of COS-1 cell in the 15cm culture dish after the transfection growth 48 hours.Also cultivated 10 minutes with the careful washed cell of PBS then with the PBS of the 25m MEDTA that contains 5ml.Isolated cell from the flat board once and again is suspended in the 5ml buffer reagent to measure cell count with binding buffer agent (DMEM, 25mMHEPES, pH7.5,0.15% gelatinum) washing then.Use 10PM at 15 ℃ 125I-VEGF cultivates this cell suspending liquid 90 minutes with the 500ml cumulative volume, and the concentration that improves unmarked part is (by 0-7 * 10 -9), by part in the conditioned medium of the COS-1 cell of temporary transient expression VEGF expression with they (164 amino acid form of purifying; Breier et al., 1992).After the cultivation, wash these cells with cold 0.1%PBS.Free ligand is removed with being suspended in the binding buffer agent again by the repeated centrifugation separation.At last, measure and this cell bonded with gamma counter (Riastar) 125On radioactivity.With Munson, P.J. and Rodbard, the method for D. (1980Anal.Biochem.107: 220-235) analyze the gained data.(11) with the retroviral vector of the negative mutant code of Flk-1 trans-dominant
With the recombinant chou retroviral vector construct become to contain the amino acid whose coding region of Flk-1 acceptor 1-806 (pLX Flk-1c1.1 and c1.3, Figure 12).The recombinant virus that use contains truncate c-fms acceptor mutant (pNTK cfms TM c1.7) in contrast.For obtaining celliferous virus, contain viral conditioned medium infecting mouse GPE cell with the both sexes (amphotrophic) that are the PA317 cell of the retroviral DNA transfection of recombinant chou.Or separately implantation of C6 glioblastoma tumour cell or celliferous virus are implanted in the nude mice together jointly.Below pointed out the cell count of two groups of test injections.When tumour occurs first, measured gross tumor volume to obtain growth curve in every 2-3 days.
Test NO 1
Figure 93115345X_IMG1
2. result
(1) separation of Flk-1
Be to differentiate the RTK that during mice develop, is expressed, carried out using the PCR in two kinds of degeneracy oligonucleotide primer storehouses of designing according to the conservative sequences of PTK kinases district inner height to measure (Hanks, S.K.et al., 1988, Science 241: 42-52).The DNA(Fahrner that use is extracted from the λ gt10 cDNA of 8.5 days mice develop stages mice embryonic of being in many atomizations and beginning, K.et al., 1987, EMBO.J., 6: the 1497-1508) template in measuring as PCR.Regulate angiopoietic RTK in order to differentiate, use similar primer to strengthen RTK cDNA sequence with similar method from capillary endothelial cells, this cell is the (Robertson that separated in the moment of brain endothelial cell hyperplasia maximum the mouse brain in 4-8 days from the back of being born, P.L.et al., 1985, Devel.Brain Res.23: 219-223).Two kinds of methods all produce the tire liver RTK that will address recently, cDNA sequence (Figure 11 .SEQ.ID NO. :) (Matthews.W.et al., 1991, the Proc.Natl.Acad.Sci.U.S.A.88: 9026-9030) of Flk-1 coding.According to amino acid identity, this acceptor is a member (Ullrich in the RTK III type subclass, A.and Schlessinger, J.1990, Cell 61: 203-212) and closely related with human body flt, it also contains seven kinds of immunoglobulins replicas in its zone, extracellular, other RTK of according to that subtribe only contains 5 kinds of this class replicated architecture (Matthews with it, W.et al., 1991, Proc.Natl.Acad Sci.U.S.A. 88: 9026-9030).Flk-1 and KDR(Terman, B.I.et al., 1991, Oncogene 6: 1677-1683) and TKr-C(Sarzani, R.et al., 1992, Biochem.Biophys.Res.Comm.186: 706-714) relatively, show that these are respectively people and the rat homologue (Fig. 1) of FIK-1.
(2) expression of Flk-1 mRNA during fetal development
As the first step of illustrating the Flk-1 biological function, in the mice embryonic of different developmental phases, analyze the expression of Flk-1 mRNA.The volume expression of main 5.5KbmRNA between 9.5 days and 18.5 days has been pointed out in the test of Northern blot hybridization, and when finishing near gestation, significantly descend (Fig. 2 A).After birth, find in 4-8 days the brain kapillary to compare Flk-1mRNA highly enriched (Fig. 2 B) with total brain mRNA.
Carry out in situ hybridization to obtain the relevant more detailed information that Flk-1 expresses in different embryo stages.Use a kind of strand antisense that contains Flk-1 extracellular region territory, the dna probe of 261P Nucleotide length is as probe, because it produces the most special hybridization signal.As an example, 14.5 days embryos' parallel arrow shape part has been shown among Fig. 3.Measure high-caliber hybridization at ventricle, lung and meninx; Its hetero-organization contains less expression Flk-1mRNA cell as manifesting in brain, liver and the maxilla.In the internode district of vertebra and also observed F1k-1 at atrium and aortal internal surface place and expressed thin chain.Shown that than the high power amplification as if the expression of F1k-1 be limited to kapillary.For example, more approaching heart film inspection shows that positive signal (Fig. 4 A) is only arranged in ventricle kapillary and interior leather sheet.In lung, in peripheral segmental bronchus kapillary, measure Flk-1 and express, but in the segmental bronchus kapillary, do not measure (Fig. 4 D).Aorta demonstrates in endotheliocyte, and strong hybridization (Fig. 4 C) is not arranged in the flesh layer.
(3) expression of Flk-1 during the organ vascularization
11.5 the neural skin in it mice embryonic akrencephalon is a kind of big dimension pipe; Tie up for the first time the pipe beginning of sprouting and then radially invade organ (Bar, J., 1980.Adv.Anat.Embryol.Cell, the Biol.59: 1-62 that forms by the nervus peripheralis vascular stand; Risou, W.and Lemmon, V.1988, Dev, Biol.125: 441-450).As shown in Fig. 5 A, in this stage, enclose in the neuroplexus and in the dimension pipe bud of invading Flk-1 to express be high.These in situ hybridizations are analyzed endotheliocyte in the hyperplasia that has indicated vascular bud and have been expressed endotheliocyte in the hyperplasia of Flk-1mRNA vascular bud.At 14.5 days of the vascularization of neural outer height, a series of radial tubes of intraneural plexus and branching pipe then contained lot of F lk-1mRNA(Fig. 5 B).When sprouting and back 4 days of the birth of endotheli ocytosis when reaching the highest, in endotheliocyte, observe the strong expression (Fig. 5 C) of F1k-1mRNA.On the contrary, in the adult brain that vascularization has stopped, Flk-1 expresses very low (Fig. 5 D) and shows and mainly is limited to choroid plexus (Fig. 6), in this choroid plexus, and the cell expressing Flk-mRNA in the interior vascular lamina, and endotheliocyte is not expressed (Fig. 6 A, B).
Embryo kidney is tieed up pipeization (Ekblom, P.et al., 1982, Cell.Diff.11: 35-39) by the vascularization process.Renal glomerulus and dimension pipe kapillary and epithelium form take place to grow synchronously.In the back 4 days kidney of birth,, in predetermined renal glomerulus kapillary, observe significant Flk-1 and express (Fig. 7 A) except other extracapillary.As if this expression continue to exist (Fig. 7 C) and relatively more confirmed this expression in renal glomerulus with the kidney at birth back initial stage in the kidney of growing up.
(4) expression of the Flk-1 among the endotheliocyte my late grandfather
In order to detect may participating in of Flk-1 in the vascular development commitment, carried out embryo's analysis of different steps during blood island forms.In the arrow shape part of 8.5 days embryonic deciduas, in the parent blood vessel in this decidua, FIK is expressed in the yolk sac with in the trophectoderm and has been measured.In allantois He in the embryo, also find Flk-1 mRNA, mainly be positioned at the part (Fig. 8 A) of matter between discovery.When the high power of deciduomata is amplified, found that in the lining blood vessels of forming by endotheliocyte the Flk-1 mRNA of height expresses (Fig. 8 B).In yolk sac, the mesoderm that hybridization signal only is limited to or vascular cell breaks up therein (Fig. 8 C).Fig. 8 D has shown the blood island that high power is amplified, and wherein periphery or vascular cell have been expressed high-caliber Flk-1mRNA.
(5) Flk-1 is a kind of to the VEGF high-affinity receptor
In situ hybridization result's detailed mensuration reaches and recently by Breier, (1992, Development 114: 521-532) Bao Dao the result about those hybridization of VEGF compares the remarkable similarity that has shown expression pattern to people such as G..In addition, VEGF(Breier in the expression of Flk-1 in the renal glomerulus internal layer and the epithelial cell that centering on, G.et al., 1992, Development 114: thus 521-532) cause the possibility of secretion relation between these cell types to represent respectively that also the ligand-receptor of VEGF and Flk-1 concerns.In order to test this hypothesis, total length F1k-1cDNA is cloned among the mammalian expression vector pCMV that transcribes the contrast key element that contains the loose virus of human body cell (Gorman, C.M.et al., 1989, Virology 171: 377-385).For the transient expression of this receptor, then the Flk-1 expression plasmid is transfected into COS-1 or fibrocyte.
By particular combination crosslinked and that competed in conjunction with test determination VEGF and Flk-RTK.In order to the COS-1 cell culture of pCMV-Flk-1 expression vector transfection through purifying 125The VEGF of I mark.Taking a picture to analyze with DSS crosslinked and immunoprecipitation subsequently, PAGE and radioactive automatic developing demonstrates the about 220KD that does not record and is with in the simultaneous test with the COS-1 cell of untransfected, and may be the complex body (Fig. 9 A) of representing the VEGF/Flk-1 acceptor.In addition, VEGF with 125I-VEGF contention and with combine (Fig. 9 B) of the Flk-1 that expresses the COS-1 cell, and the COS-1 cell debond of untransfected 125I-VEGF.VEGF is specific with the interaction of acceptor on transfectional cell, as PDGF-BB not with 125The combination competition of I-VEGF.The binding data analysis has shown and has been about 10 -10M Kd, this expression Flk-1 is the high-affinity receptor of a kind of VEGF.This discovery has represented consumingly that with F1k-1 and VEGF in situ hybridization result Flk-1 is a relevant acceptor on a kind of physiology to VEGF.
Carried out automatic phosphorylation assay to confirm the biological dependency of VEGF and Flk-1 receptors bind.In the DMEM that contains 0.5% tire calf serum, make hungry 24 hours of the COS-1 cell of transient expression Flk-1, stimulate with 0.5mMVEGF, and dissolving.Make this receptor immunoprecipitation with the specific polyclonal antibody CT128 of Flk-1, analyzing also by SDS-DAGE then uses anti-phosphorus tyrosine antibody 5E2 to carry out immunoblotting (Fendly subsequently, B.M.et al., 1990, Cancer Research 50: 1550-1558).Shown in Figure 10 is that the VEGF of Flk-1 express cell stimulates the tyrosine phosphorylation that has caused significantly inducing 180KD Flk-1 acceptor.
(6) suppress tumour cell by Flk-1 trans-dominant-feminine gender
The Flk-1 acceptor is considered to play a major role in vasculation and vascularization.Thereby the activity that suppresses Flk-1 can suppress the vascularization of developmental tumour and suppress its growth.In order to test this hypothesis, with tumour cell (C6 rat glioblastoma) and produce that the retroviral mouse cell of recombinant chou with truncate Flk-1 acceptor coding mixes and subcutaneous implantation nude mice in.The C6 emiocytosis meeting of implanting is in conjunction with the VEGF that also activates the Flk-1 acceptor of expressing on the mouse endothelial cell surface.Without any angiopoietic inhibition the time, this endotheliocyte is understood hyperplasia and is shifted to tumour cell.On the other hand, if when injection, tumour cell and the collaborative injection of recombinant chou retrovirus that produces dominance-negative F1k-1 coding, then can become to the endotheliocyte of the growth of tumour cell of implanting and be infected by the recombinant chou retrovirus, this may cause dominance-negative Flk-1 mutant to be expressed and suppress endogenous Flk-1 signal.The inhibition of endotheli ocytosis and migration can cause implanted cell to become the failure of vascularization, and this will cause suppressing tumor growth.Shown in Figure 12 and 13, in accept producing the mouse of truncate Flk-1 Transplanted cells, tumor growth is suppressed significantly, and this mode that has shown that the expression of truncate Flk-1 can dominance-feminine gender plays the active effect of the endogenous wild-type Flk-1 of inhibition.
The invention is not restricted to the scope of illustrative embodiment, they are intended to illustrate the single aspect of the present invention, and any clone, DNA or the aminoacid sequence that are equal on function are all within the scope of the present invention.In fact, except this paper has stated, various changes of the present invention will be conspicuous according to above statement and accompanying drawing to the those of ordinary skill of prior art.These changes will be included within the scope of the claim that awaits the reply.
What also it is to be understood that is that all base pair sizes given to Nucleotide are approximation and are used for illustration purpose.
Figure 93115345X_IMG2
Figure 93115345X_IMG4
Figure 93115345X_IMG5
Figure 93115345X_IMG6
Figure 93115345X_IMG7
Figure 93115345X_IMG8
Figure 93115345X_IMG9
Figure 93115345X_IMG10
Figure 93115345X_IMG11
Figure 93115345X_IMG12

Claims (43)

1, a kind of recombinant DNA carrier, this carrier contain with operation go up with control the host in the relevant F1k-1 nucleotide sequence coding of adjusting sequence of genetic expression.
2, a kind of recombinant DNA carrier, this carrier contain with operation go up with control the host in the relevant Flk-1 fusion rotein nucleotide sequence coding of adjusting sequence of genetic expression.
3, a kind of genetically engineered host cell, this cell contain the recombinant DNA carrier of claim 1 or 2.
4, a kind of genetically engineered cell is, this clone contains the recombinant DNA expression vector of claim 1 and expresses Flk-1.
5, the genetically engineered cell of claim 3 system, this clone is expressed Flk-1 on this cell surface.
6, a kind of genetically engineered cell is, this clone contains the recombinant DNA expression vector of claim 2 and expresses the Flk-1 fusion rotein.
7, the genetically engineered cell of claim 6 system, this clone is expressed the Flk-1 fusion rotein on this cell surface.
8, a kind of method for preparing recombinant chou Flk-1, this method comprises:
(a) cultivate recombinant DNA expression vector transformed host cells and this host cell expression Flk-1 that uses claim 1; And
(b) reclaim the Flk-1 gene product in the cell culture thus.
9, a kind of method for preparing recombinant chou Flk-1 fusion rotein, this method comprises:
(a) cultivate the recombinant DNA expressing gene transformed host cells of using claim 2, and this host cell expression F1k-1 fusion rotein, and
(b) by reclaiming the Flk-1 fusion rotein in this cell culture.
10, a kind of separated recombinant chou Flk-1 receptor protein.
11, a kind of warm albumen, this albumen comprise the Flk-1 that connects foreign protein or peptide sequence.
12, a kind of oligonucleotide, this Nucleotide will compensate the antisense sequences coding on the part of Flk-1 nucleotide sequence, and suppress Flk-1 gene translating in cell.
13, the oligonucleotide of claim 12, this Nucleotide compensation is on the nucleotide sequence coding with Flk-1 N-terminal district.
14, a kind of monoclonal antibody, this antibody mediated immunity specifically combine with the epitope of Flk-1.
15, the monoclonal antibody of claim 14, this antibody competition ground inhibition VEGF combines with Flk-1's.
16, the monoclonal antibody of claim 14, this antibody is connected on the cytotoxic agent.
17, the monoclonal antibody of claim 14, this antibody is connected on the radio isotope.
18, the method for a kind of screening and discriminating VEGF antagonist, this method comprises:
(a) clone that will express Flk-1 when VEGF is arranged contacts with test compound; And
(b) measure combination and the cytosis whether this test compound is suppressed at the VEGF on this clone,
Wherein, this antagonist is to be suppressed at the combination of the VEGF on the clone and the compound of cytosis is differentiated as those.
19, the method for a kind of screening and discriminating VEGF agonist, this method comprises:
(a) clone that will express Flk-1 when existing or not having VEGF contacts with test compound;
(b) whether test compound suppresses combining of VEGF and this clone when having VEGF; And
(c) determine whether test compound does not simulate the cytosis of the VEGF on clone when having VEGF, wherein this agonist is as being suppressed at the combination of the VEGF on the clone, but those test compounds of the cytosis of simulation VEGF on clone and being differentiated.
20, claim 18 or 19 method, the clone that this clone is genetically engineered.
21, claim 18 or 19 method, the wherein endogenous expression of this clone Flk-1.
22, the method for a kind of screening and discriminating VEGF antagonist, this method comprises:
(a) Flk-1 albumen is contacted with random peptide library so that F1k-1 discerns and combines with one or more peptide kinds in the storehouse;
(b) combination of separation Flk-1/ peptide;
(c) isolating peptide sequence among the determination step c; And
(d) whether the determination test compound suppresses combination and the cytosis of VEGF, and wherein this antagonist is to suppress being differentiated in conjunction with the peptide of cytosis of VEGF as those.
23, the method for a kind of screening and discriminating VEGF agonist, this method comprises:
(a) Flk-1 albumen is contacted with random peptide library so that Flk-1 identification and in conjunction with one or more peptide kinds in the storehouse;
(b) combination of separation Flk-1/ peptide;
(c) isolating peptide sequence among the determination step c; And
(d) measure the cytosis whether this peptide simulates VEGF when not having VEGF, wherein this agonist is to suppress combination as those, but the peptide of simulation Flk-1 cytosis and being differentiated.
24, claim 22 or 23 method, wherein Flk-1 albumen has been genetically engineered.
25, a kind of method of regulating the endogenous enzyme activity of Tyrosylprotein kinase Flk-1 acceptor in the Mammals, this method comprise that part to the Flk-1 receptor protein of a kind of significant quantity of administration is with the regulatory enzyme activity.
26, the method for claim 25, wherein the part of Flk-1 acceptor is VEGF.
27, the method for claim 25, wherein the part of Flk-1 acceptor is the VEGF agonist.
28, the method for claim 25, wherein the part of Flk-1 acceptor is the VEGF antagonist.
29, the antagonist of claim 28, this antagonist are that a kind of immunity is specifically in conjunction with the monoclonal antibody of Flk-1 epitope.
30, the antagonist of claim 28, this antagonist are a kind of solubility Flk-1 acceptors.
31, the method for claim 25, wherein the enzymic activity of receptor protein is enhanced.
32, the method for claim 25, wherein the enzymic activity of receptor protein is lowered.
33, the method for claim 31, wherein ligand stimulation endotheli ocytosis.
34, the method for claim 32, wherein part suppresses endotheli ocytosis.
35, the method for claim 32, wherein part suppresses vascularization.
36, a kind of recombinant vector, this carrier contain and will have the VEGF of inhibition in conjunction with the active truncate Flk-1 nucleotide sequence coding of the dominance-feminine gender of cytosis.
37, the recombinant vector of claim 36, this carrier contain the 1-806 amino acid nucleotide sequence coding with Flk-1.
38, the recombinant vector of claim 36, wherein this carrier is a kind of retroviral vector.
39, the recombinant vector of claim 38, this carrier contain the 1-806 amino acid nucleotide sequence coding with Flk-1.
40, a kind of genetically engineered cell is, this clone contains the recombinant DNA carrier of claim 36 and expresses truncate Flk-1.
41, a kind of genetically engineered cell is, this clone contains the recombinant vector of claim 38 or 39 and produces the retroviral infection particle of expressing truncate Flk-1.
42, it is active that the truncate Flk-1 receptor protein of a kind of separated recombinant chou, this albumen have the dominance-feminine gender that suppresses VEGF bonded cytosis.
43, a kind of method that is adjusted in VEGF cytosis in the Mammals, this method comprise the truncate Flk-1 receptor protein to the inhibition VEGF bonded cytosis of a kind of significant quantity of this administration.
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